Alkylguanyltransferase Assays

ABSTRACT

This invention provides a method to determine alkylguanyltransferase activity in a sample, comprising steps of placing the sample in an appropriate condition so that the AGT is functional; contacting the sample with an AGT Detector under conditions permitting the binding of AGT and AGTD to produce a signal; and measuring the signal, thereby determining the AGT activity in said sample. This invention provides different uses of this method.

This application is a continuation of U.S. Ser. No. 11/373,623, filedMar. 9, 2006, now U.S. Pat. No. 7,273,714, issued Sep. 25, 2007, whichclaims the benefit of U.S. Ser. No. 60/663,454, filed Mar. 18, 2005 andU.S. Ser. No. 60/660,738, filed Mar. 11, 2005. The contents thepreceding applications are hereby incorporated herein by reference intheir entireties.

Throughout this application, various references are referred to anddisclosures of these publications in their entireties are herebyincorporated by reference into this application to more fully describethe state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

Chemotherapeutic agents that alkylate the O⁶ position of guanine in DNAsuch as Carmustine (Ishibashi, et al., J. Biol. Chem., 269: 7645-7650,1994) 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, fotemustine,dacarbazine, streptozotocin, procarbazine, and temozolomide (TMZ) areused primarily to treat brain cancer, melanoma, lymphoma, andgastrointestinal cancers. The effectiveness of these agents, however, islimited by alkylguanyltransferase (AGT), a protein that repairsO⁶-alkylguanine adducts and is up-regulated in several tumors duringprogression (Ishibashi et al., Mutat. Res., 315: 199-212, 1994; Citronet al., Cancer Investig., 12: 605-610, 1994; Kokkinakis et al., CancerRes., 57: 5360-5368, 1997).

Furthermore, selection of resistant AGT phenotypic populations aftertreatment with alkylating agents seems to be the reason for therecurrence of tumors of even a more resistant phenotype (Lage et al., J.Cancer Res. Clin. Oncol., 125: 156-165, 1999). Tumor resistance to DNAalkylation could be theoretically reversed with AGT inhibitors thatreact with and inactivate the protein. Despite considerable advances inthis field, methodologies to sensitize tumors by depleting AGT and theselection of the appropriate chemotherapeutic agent to be combined withAGT depleting drugs are still under evaluation.

An additional important issue in combining DNA alkylating agents withAGT inhibitors is whether to include such inhibitors in the treatment oftumors with no or low AGT content, especially because such a combinationlimits the dose of the alkylating agent. Dose is important for severalreasons, including the fact that the alkylating agent itself mightquench low levels of AGT. A case in point is TMZ, which at a dose of 100mg/kg eliminates all of the AGT activity in tumors having moderate AGTlevels for a prolonged time period (Chinnasamy et al., Blood, 89:1566-1573, 1997). In addition, the inverse correlation between AGTlevels and effectiveness of BCNU against central nervous system tumors(Belanisch et al., Cancer Res., 56: 783-788, 1996; Jaeckle et al., J.Clin. Oncol., 16: 3310-3315, 1998) suggest that there may be no benefitin treating AGT-deficient tumors with AGT inhibitors. Determining thethreshold of AGT activity that could be overcome by alkylating agentswithout the use of AGT inhibitors may be beneficial.

High AGT activity confers resistance to DNA alkyating agents (seeabove). A sensitive and fast turn-round AGT assay could be used toselect patients with low AGT activities, and thus provide a betteroutcome for patients receiving DNA alkylating agents. A few assays havebeen developed but they are either not sensitive enough or solabor-intensive that they are not suitable for routine laboratory use(Wu et al., Cancer Res., 47: 6229, 1987; Gerson et al., J. clin.Invest., 76:2106, 1985; Kreklau et al., Nucleic Acid Res., 29:2558,2001).

This invention provides an improved assay, which is simple andefficient, for alkylguanyltransferase.

SUMMARY OF THE INVENTION

This invention provides a method to determine alkylguanyltransferase(AGT) activity in a sample, comprising steps of:

-   -   (a) placing the sample in an appropriate condition so that the        AGT is functional;    -   (b) contacting the sample with an alkylguanyltransferase        detector (AGTD) under conditions permitting the binding of AGT        and AGTD to produce a signal; and    -   (c) measuring the signal, thereby determining the AGT activity        in said sample.

This invention provides an AGTD which contains:

-   -   (a) a AGT binder; and    -   (b) a detectable signal; and optionally,    -   (c) a system that detect the signal which subsequently generates        a second signal.

The first or second signal, in the form of color, light, fluorescence,or radioactivity, is then detected with the corresponding methods.

Finally, this invention provides different uses of the above method.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1. Detection of AGT activity

FIG. 2. Signal molecule conjugated to EX

FIG. 3. Signal molecule conjugated to AP

FIG. 4. Signal molecule conjugated to O6BG

FIG. 5. Scintillation Proximity Assay

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a method to determine AGT activity in a sample,comprising steps of:

-   -   (a) placing the sample in an appropriate condition so that the        AGT is functional;    -   (b) contacting the sample with an AGTD under conditions        permitting the binding of AGT and AGTD to produce a signal; and    -   (c) measuring the signal, thereby determining the AGT activity        in said sample.

In an embodiment, the sample is from a patient. As used herein, AGTDcomprises:

-   -   (a) a AGT binder;    -   (b) a detectable signal; and optionally,    -   (c) a system that detect the signal which subsequently generates        a second signal.

AGTD may carry more than one signal. The signal may be detected directlyor indirectly. For indirect detection, other agent(s) may be used tofacilitate said detection. For example, the detectable signal mayinclude an antibody which would be recognized by a second antibody. Thesecond antibody is linked to a marker which is detected by standardmethods.

The current assay employs an agent (AGT binder) that binds to AGT, whichis capable of transferring a chemical moiety from the AGT binder to AGT.The AGT binder can be an agonist, antagonist, activator, or inhibitor ofAGT. These agents include but not limit to N¹, O⁶-ethanoxanthosine (EX)(Noll and Clarke, Nucleic Acid Res., 29: 4025, 2001),2′-deoxy-6-(cystamine)-2-aminopurine (AP) (Paalman et al., Nucleic AcidRes., 25:1795, 1997), temozolomide, benzylguinine, O(6)-Benzylguanine(O6BG), 8-aza-O(6)-benzylguanine, O(6)-(4-bromophenyl)-guanine (O6BTG),O(6)alkylguanine, and analogues of these agents. AGT binder alsoincludes DNA or oligoribonucleotide or oligodeoxyribonucleotide(collectively called oligonucleotide) containing the above agents.

In one embodiment, the chemical moiety transferred from AGT binder toAGT contains a signal such as radioactivity, fluorescence, luminescence,or electro-spin resonance (ESR). The signal transferred from AGT binderto AGT is directly determined by various methods according to the signaltransferred. In another embodiment, the chemical moiety transferred toAGT is indirectly detected with an immunoassay or other affinity bindingassay (FIG. 1).

The AGT binder includes an inhibitor or activator of AGT. In anembodiment, the AGT binder is EX and the AGTD is biotin-conjugated EX(FIG. 2). In another embodiment, the AGT binder is AP and the AGTD isbiotin-conjugated AP (FIG. 3). In yet other embodiment, the AGT binderis 06BG and the AGTD is biotin-conjugated 06BG (FIG. 4).

In a separable embodiment, the detectable signal molecule is afluorophore.

The invention also provides a method to determine AGT activity, whichcomprises: a) AGT, b) an AGT binder carrying a radioactive moleculewhich is transferred to AGT, and Scintillation Proximity Assay (SPA)bead that binds AGT directly or indirectly (FIG. 4). In a furtherembodiment, the AGT binder is radioactive-labeled EX or AP.

The invention provides a method to determine AGT activity, comprising ofAGT and an AGT binder carrying a chemical moiety which is transferred toAGT and the chemical moiety transferred to AGT is detected bystreptavidin or an antibody.

This invention provides AGTDs which are not previously known.

This invention also provides a kit with a compartment containing AGTD.

This invention will be better understood from the examples which follow.However, one skilled in the art will readily appreciate that thespecific methods and results discussed are merely illustrative of theinvention as described more fully in the claims which follow thereafter.

EXAMPLE 1 Scintillation Proximity Assay (SPA)

SPA beads are microscopic beads contain a scintillant that can be exciteby radioactive signals to emit light. This excitation event occurs whenradiolabeled molecules of interest are bound to the surface of the bead,either directly or indirectly (see FIG. 5); the light emitted can bedetected with a scintillation counter.

Preparation of reagents. EX, labeled with 3[H] at various positions atthe guanine moiety is used as the AGT binder. The AGT-specificmonoclonal antibody (clone MT 3.1) (NeoMarkers, Fremont, Calif.) is usedto capture AGT. SPA beads coated with anti-mouse antibody is used tobind the MT3.1 anti-AGT antibody. The guanine moiety containing 3[H] istransferred from the AGT binder to anti-AGT antibody and subsequently iscaptured by SPA beads.

Assay. Samples containing AGT, undiluted or diluted up to 100×, is addedto a buffer containing a) 3[H]EX (1 fmol to 100 nmol), anti-AGT antibody(MT 3.1, 1 ng to 1 ug), and anti-mouse SPA bead (e.g. antimouse YSibead, 1 ug to 10 mg, Amershan Bioscience). SPA beads coated with ProteinA or Protein G can also be used. The solution is incubated for 15 to 60minutes and radioactivity is determined with a scintillation counter.AGT activity is presented as dpm per mg protein. A précised AGT unit isdetermined by using 3[H]EX standard for constructing a standard curve.In this case, AGT activity is defined as fmol EX per mg protein. AGTactivity can also be expressed as per DNA or cell number basis. Analternative method is to use biotin labeled anti-AGT antibody andstreptavidin coated SPA beads.

EX can also be labeled with other radioactive isotopes or at otherpositions.

EXAMPLE 2 Immunoassay

This assay is based on using two antibodies or one antibody andstreptavidin; one captures AGT and the other detects the chemical moietytransferred to AGT.

Preparation of reagents. The AGT-specific monoclonal antibody (clone MT3.1) (NeoMarkers, Fremont, Calif.) is used to coat a 96-well plate tocapture AGT. Streptavidin-conjugated peroxidase is used to detect thechemical moiety transferred to AGT.

Assay. This assay is based on a sandwich Elisa assay (Chapter 14,Antibodies, A laboratory Manual, Harlow and Lane, Cold Spring HarborLaboratory, 1988). Samples containing AGT, undiluted or diluted up to1000×, is added to a buffer containing biotin-conjugated EX (1 fmol to100 nmol). The solution is incubated for 15 to 60 minutes; theincubation is stopped by the addition of a buffer with or without adetergent (e.g. 0.1% SDS or 0.1% NP40). The solution is added to theplate that coated with anti-AGT antibody and further incubated for 15 to60 minutes. The plate is washed three times with a buffer containing amild detergent. Streptavidin-conjugated peroxidase, at various dilutionis added to the well and incubated for 15 to 60 minutes. The amount ofbiotin-conjugated EX bound to the AGT is determined by standard Elisamethods. AGT activity is presented as O.D. per mg protein. A précisedAGT unit is determined by using biotin-conjugated EX standard forconstructing a standard curve. In this case, AGT activity is defined asfmol EX per mg protein. AGT activity can also be expressed as per DNA orcell number basis.

EXAMPLE 3 Fluorescence Assay

The chemical moiety transferred from AGT binder to AGT contains afluorophore (e.g. fluorescein, Texas red; Handbook of fluorescent probesand research products (P.62 and P15, Haugland, 9^(th) ed., MolecularProbes, Eugene, Oreg.).

Preparation of the AGTD. A fluorescein molecule (Molecular Probe) isused to conjugate to the 8- or 9-position of the guanine moiety of EX(FIG. 3).

Assay. Samples containing AGT, undiluted or diluted up to 1000×, isadded to the AGTD, at concentrations from 1 fmol/mg protein to 10000fmol/mg protein. Molar ratio of AGT to AGTD is ranging from 1 to 1000,preferably from 5 to 200. AGT is incubated with AGTD in a buffer thatmaximize AGT activity. AGT activity is determined with a fluorescencespectrophotometer. For fluorescein, the excitation and emissionwavelengths are 494 nm and 518 nm, respectively. AGT activity ispresented as fluorescence unit per mg protein. A précised AGT unit isdetermined by using EX conjugated with fluorescein as standard forconstructing a standard curve. In this case, AGT activity is defined asfmol EX per mg protein. AGT activity can also be expressed as per DNA orcell number basis.

Other detecting method such as fluorescence polarization ortime-resolved fluorescence spectroscopy can also be used.

1. A method to determine AGT activity, comprising AGT and an AGT bindercarrying a signaling molecule which is transferred to AGT.
 2. The methodaccording to claim 1 in which the AGT binder is an inhibitor of AGT. 3.The method according to claim 1 in which the AGT binder is an activatorof AGT.
 4. The method according to claim 1 in which the AGT binder isEX.
 5. The method according to claim 1 in which the AGT binder is anoligonucleotide containing EX.
 6. The method according to claim 1 inwhich the signal molecule is a fluorophore.
 7. The method according toclaim 1 in which the signal molecule is biotin.
 8. A method to determineAGT activity, comprising: a) AGT, b) an AGT binder carrying aradioactive molecule which is transferred to AGT, and SPA bead thatbinds AGT directly or indirectly.
 9. The method according to claim 8 inwhich the AGT binder is radioactive-labeled EX.
 10. The method accordingto claim 1, set forth in FIG.
 1. 11. A method to determine AGT activity,comprising of AGT and an AGT binder carrying a chemical moiety which istransferred to AGT and the chemical moiety transferred to AGT isdetected by streptavidin.
 12. The Scintillation Proximity Assay for AGT,set forth in FIG.
 5. 13. An AGT binder, not previously known.
 14. An AGTbinder, set forth in FIG. 3 or
 4. 15. A compound which binds to AGT,comprises an AGT binder and biotin.
 16. The compound according to claim15 wherein the AGT binder is EX.
 17. A composition comprising thecompound of claim 15 or 16.